Whole cell lysates were boiled for 10 min in 4× NuPAGE LDS loading buffer (Invitrogen) and cooled on ice. Samples were loaded onto precast 4–12% NuPAGE Bis‐Tris gels and transferred onto PVDF membranes (Hybond‐P, Amersham Biosciences). Blocked membranes were probed using rabbit anti‐MxA (Abnova) or mouse anti‐β‐actin (Sigma) antibodies. For fluorescent detection the blocking step and all antibody incubations were performed in LI‐COR blocking buffer. Membranes were allowed to dry and proteins were detected using the Odyssey Western Blot Scanner (LI‐COR).
Odyssey western blot scanner
Manufactured by LI COR
The Odyssey Western Blot Scanner is a high-performance imaging system designed for the detection and quantification of proteins on Western blots. It uses near-infrared fluorescence technology to provide sensitive and linear detection of proteins across a wide dynamic range.
Automatically generated - may contain errors
Lab products found in correlation
Mini protean tgx precast protein gel, by Bio-Rad (2 mentions)
Irdye 680rd donkey anti rabbit igg, by LI COR (2 mentions)
Irdye 800cw goat anti mouse igg, by LI COR (2 mentions)
Nupage lds loading buffer, by Thermo Fisher Scientific (1 mentions)
Mouse anti β actin, by Merck Group (1 mentions)
Hybond p, by Cytiva (1 mentions)
4 protocols using odyssey western blot scanner
1
Western Blot Analysis of MxA Protein
2
Western Blot Quantification Protocol
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Cells were lysed in RIPA buffer at 3×105/50 μL density. Protein concentrations were determined using the Bradford protein assay. Laemmli buffer was added to protein lysates at a 1:1 ratio. The lysates were loaded onto 4–15% Mini-PROTEAN® TGX™ Precast Protein Gels (Cat# 4561086, Bio-Rad, Hercules, CA), and proteins were subsequently transferred onto a polyvinylidene fluoride membrane. The membrane was blocked with 5% milk in 0.05% Tween-20 in PBS (PBS-T) to prevent nonspecific binding of antibodies. Primary antibody incubation was performed in PBS-T with 1% milk at 4°C overnight (refer to S1 Table for list of primary antibodies used). IRDye® 680RD donkey anti-rabbit IgG or IRDye® 800CW goat anti-mouse IgG (LI-COR Biosciences®, Lincoln, NE) was incubated with the membranes for 1 hour at room temperature in PBS-T with 1% milk. The membranes were washed 3 times with PBS-T and scanned using an Odyssey Western blot scanner (LI-COR Biosciences®). All protein quantification was performed using LI-COR image analysis software.
3
Immunoprecipitation and Western Blot Analysis
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Cells (at least 1.5 × 107) were harvested and lysed within ice-cold RIPA (150 mM NaCl, 50 mM Tris pH7.6, with 1 mM PMSF, 1 g/ml leupeptin, 1 g/ml aprotinin, and 1 g/ml pepstatin) for 30 min. The lysates were centrifuged at 12,000 g for 5 min at 4°C to remove cell debris. Five percent of the supernatant was used as input. The remaining supernatant was then incubated with the indicated primary antibody or IgG at 4°C with rotation overnight. Next day, the supernatants were incubated with 35 mL protein A/G Sepharose beads at 4°C with rotation for another 3 hours. For immunoblotting assays, the immunoprecipitates were boiled in 6x SDS loading buffer. The protein was separated by SDS-PAGE and transferred to nitrocellulose membrane for immunoblotting with primary antibodies or IgG and appropriate secondary antibodies. The membrane was scanned with an Odyssey western blot scanner (LI-COR Biosciences).
4
Western Blot Protein Quantification
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Cells were lysed in RIPA buffer at 310 5 /50 µL density. Protein concentrations were determined using Bradford protein assay. Laemmli buffer was added to protein lysates at a 1:1 ratio. The lysates were loaded onto 4-15% Mini-PROTEAN® TGX™ Precast Protein Gels (Cat# 4561086, Bio-Rad, Hercules, CA), and proteins were subsequently transferred onto a polyvinylidene . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (which this version posted July 10, 2020. ; https://doi.org/10.1101/2020.07.10.197178 doi: bioRxiv preprint 6 fluoride (PVDF) membrane. The membrane was blocked with 5% milk in PBS-T (0.05% Tween-20 in PBS) to prevent nonspecific binding of antibodies. Primary antibody incubation was performed in PBS-T with 1% milk at 4°C overnight (refer to Supplementary Table 1 for list of primary antibodies used). IRDye® 680RD donkey anti-rabbit IgG or IRDye® 800CW goat anti-mouse IgG (LI-COR Biosciences ® , Lincoln, NE) was incubated with the membranes for 1 hour at room temperature in PBS-T with 1% milk. The membranes were washed 3 times with PBS-T and scanned using an Odyssey western blot scanner (LI-COR Biosciences ® ). All protein quantification was performed using LI-COR image analysis software.
The copyright holder for this preprint (which this version posted July 10, 2020. ; https://doi.org/10.1101/2020.07.10.197178 doi: bioRxiv preprint 6 fluoride (PVDF) membrane. The membrane was blocked with 5% milk in PBS-T (0.05% Tween-20 in PBS) to prevent nonspecific binding of antibodies. Primary antibody incubation was performed in PBS-T with 1% milk at 4°C overnight (refer to Supplementary Table 1 for list of primary antibodies used). IRDye® 680RD donkey anti-rabbit IgG or IRDye® 800CW goat anti-mouse IgG (LI-COR Biosciences ® , Lincoln, NE) was incubated with the membranes for 1 hour at room temperature in PBS-T with 1% milk. The membranes were washed 3 times with PBS-T and scanned using an Odyssey western blot scanner (LI-COR Biosciences ® ). All protein quantification was performed using LI-COR image analysis software.
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